JP2013535986A - Nucleic acid capture and sequencing - Google Patents

Abstract

  A method for capturing and sequencing a target nucleic acid molecule is provided. A method for determining the methylation status of genomic DNA is also provided.

Description

(Related application)
This application claims priority under 35 USC 119 (e) to US Provisional Patent Application No. 61 / 402,350 filed on August 27, 2010, the contents of which are hereby incorporated by reference. Incorporated in the description.

(Field of Invention)
The present invention relates to separating target nucleic acid molecules from a mixture of nucleic acids and determining the sequence of such molecules.

  Since the complete decoding of the human genome has been completed, genomic research has turned to “re-sequencing”, where variants such as disease-related mutations are identified throughout the genome. Re-sequencing of “segmented” genomes enriched for specific regions of interest, such as exons, requires multiple steps for “capturing” and sequencing those regions. For example, in one microarray method, genomic DNA is sheared into a specific range of size fragments, the fragments are end-repaired, ligated to specific adapters and amplified, and the amplified fragment is then subjected to a reference genomic sequence of interest. Using a microarray containing complementary probes, the captured (hybridized) fragments are eluted and amplified, and the amplified fragments are used, for example, using “next generation” sequencing techniques or resequencing arrays. Sequenced. See, for example, WO 2008/115185, Okou et al. (2007) Nature Methods 4: 907-909, Hodges et al. (2007) Nature Genetics 39: 1522-1527, and the like. If the steps required to isolate and sequence the nucleic acid of interest are reduced, efficiency and accuracy will increase, potentially reducing costs. The present invention fulfills this need and provides other advantages.

  Cytosine methylation generally occurs at genomic CpG dinucleotides and plays an important role in gene regulation and epigenetic inheritance. Certain existing methods for determining the methylation status of genomic regions use bisulfite treatment. In these methods, when denatured genomic DNA is exposed to bisulfite ions, cytosine is deaminated to uracil, while methylated cytosine is protected from this conversion. The presence or absence of a conversion phenomenon can be detected, for example, by next generation sequencing or using a probe array. See e.g. WO 2010/085343. Such methods often take multiple steps, such as amplification, capture, elution, and sequencing of bisulfite-treated DNA, or investigate the presence of conversion events in all targeted genomic regions. It is necessary to design a probe for this purpose, but it may be expensive and complicated. Further, in such a method, the methylation state at the individual DNA molecule level is not evaluated, but a group of nucleic acid molecules for a specific genomic region of interest is investigated. The present invention provides an efficient and accurate method for assessing the methylation status of genomic DNA, meeting the needs in the art and providing other benefits.

  In one aspect, a method for capturing and sequencing a target nucleic acid molecule is provided, the method comprising: (a) exposing an individual support to a nucleic acid mixture comprising the target nucleic acid molecule under hybridizing conditions, wherein the target nucleic acid molecule Forming a specific hybridization complex with a primer immobilized on an individual support in a structure capable of priming, (b) separating unbound and nonspecifically bound nucleic acids from the individual support; (c) Determine the nucleic acid sequence of the target nucleic acid molecule by exposing the solid support to polymerase and nucleotides under polymerization conditions and (d) detecting nucleic acid polymerization from immobilized primers by the polymerase using the target nucleic acid molecule as a template. Including doing.

  In one embodiment, the target nucleic acid molecule is derived from a genomic DNA region. In one such embodiment, the target nucleic acid molecule includes all or part of exons. In another embodiment, the target nucleic acid molecule is RNA, the polymerase is reverse transcriptase, and the primer comprises a 3 'poly-T sequence. In another embodiment, the target nucleic acid molecule is DNA and the polymerase is a DNA polymerase. In another embodiment, the nucleotide is labeled on the terminal phosphate. In one such embodiment, the polymerase is labeled with a FRET donor and the nucleotide is labeled with a FRET acceptor. In one such embodiment, the FRET donor is a fluorescent nanoparticle.

  In another aspect, a method for determining the methylation status of a genomic DNA fragment is provided, the method comprising (a) fixing the genomic DNA to an individual support and (b) using the fixed genomic DNA fragment as a template. By detecting the nucleic acid polymerization by the polymerase to determine the nucleic acid sequence of the DNA fragment immobilized on the solid support, (c) subjecting the immobilized genomic DNA fragment to bisulfite treatment, and (d) the immobilized By detecting nucleic acid polymerization by polymerase using a bisulfite-treated genomic DNA fragment as a template, the nucleic acid sequence of the bisulfite-treated genomic DNA fragment immobilized on an individual support is determined. , (E) comparing the nucleic acid sequence determined in (b) with the sequence determined in (d), wherein the conversion of cytosine residues of the genomic DNA fragment comprises This indicates that the residue of the genomic DNA fragment was not methylated before bisulfate treatment, and that the cytosine residue of the genomic DNA fragment was not converted indicates that the residue of the genomic DNA fragment before bisulfite treatment Indicates that was methylated.

  In one embodiment, the genomic DNA fragment is fixed to the individual support by an adapter. In one such embodiment, the adapter includes a primer binding site and cytosine in the primer binding site is protected. In one such embodiment, the polymerase of (b) and / or (d) polymerizes the nucleic acid strand from the primer annealed to the primer binding site. In another embodiment, the nucleic acid polymerization of (b) and / or (d) is detected by detecting the incorporation of labeled nucleotides. In one such embodiment, the labeled nucleotide is labeled on its terminal phosphate. In one such embodiment, the polymerase of (b) and / or (d) is labeled with a FRET donor and the nucleotide is labeled with a FRET acceptor. In one such embodiment, the FRET donor is a fluorescent nanoparticle.

FIG. 1 shows the direct capture of sheared DNA selection regions on an oligonucleotide array using complementary oligonucleotides and direct addition of bases (sequences) and polymerase during real-time DNA synthesis. FIG. 5 shows direct sequencing of captured DNA using a free 3 ′ end of an oligonucleotide used for DNA capture and labeled polymerase and labeled dNTPs allowing monitoring. FIG. 2 shows the direct capture of sheared DNA selection regions using beads coated with complementary oligonucleotides, subsequent bead placement, and added bases (sequences) during real-time DNA synthesis. Figure 2 shows the free 3 'end of an oligonucleotide used for DNA capture and captured DNA sequencing using labeled polymerase and labeled dNTP, allowing direct monitoring of the polymerase. FIG. 3 shows the direct capture of RNA on a poly-dT oligonucleotide array and conversion of the captured RNA into cDNA using the free 3 ′ end of the poly-dT oligonucleotide array and reverse transcriptase. It is a figure which determines the arrangement | sequence of the capture | acquired RNA. After cDNA conversion, the cDNA is sequenced using poly-A primers and labeled polymerase and labeled dNTP, allowing direct monitoring of added base (sequence) and polymerase during real-time DNA synthesis. Is done. Alternatively, the captured RNA uses labeled reverse transcriptase and labeled dNTP to allow direct monitoring of the added base (sequence) and reverse transcriptase during real-time DNA synthesis to obtain its sequence. And can be sequenced directly. FIG. 4 shows the direct capture of RNA using poly-dT oligonucleotides immobilized on beads, where the beads are placed on the surface and the captured RNA is then freed from the poly-dT oligonucleotide 3 'Convert to cDNA using end and reverse transcriptase. After cDNA conversion, the cDNA is sequenced using poly-A primers and labeled polymerase and labeled dNTP, allowing direct monitoring of added base (sequence) and polymerase during real-time DNA synthesis. Is done. Alternatively, the captured RNA uses labeled reverse transcriptase and labeled dNTP to allow direct monitoring of the added base (sequence) and reverse transcriptase during real-time DNA synthesis to obtain its sequence. And can be sequenced directly. FIG. 5 shows the nucleic acid methylation status by sequencing the same DNA molecule on the array in a continuous reaction, where the second round of sequencing is performed after bisulfite treatment allowing uracil conversion of methylated cytosine. It is a figure to determine. Appropriate primers, labeled polymerase and labeled dNTP allow direct monitoring of the added base (sequence) and polymerase during real-time DNA synthesis. FIG. 6 shows the nucleic acid methylation status by sequencing the same DNA molecule on the beads in a continuous reaction, where the second round sequencing is performed after bisulfite treatment allowing uracil conversion of methylated cytosine. It is a figure to determine. Appropriate primers, labeled polymerase and labeled dNTP allow direct monitoring of the added base (sequence) and polymerase during real-time DNA synthesis.

(I definition)
“Bisulfite treatment” refers to exposing a nucleic acid to bisulfite ions (eg, magnesium bisulfite or sodium bisulfite) at a concentration sufficient for uracil conversion of unprotected cytosine. “Bisulfite treatment” also refers to exposing the nucleic acid at an appropriate concentration to other reagents that may be used to convert unprotected cytosine to uracil, such as disulfite and bisulfite. “Bisulfite treatment” generally involves exposing a nucleic acid to a bisulfite ion or other reagent followed by a base such as NaOH.

  “Conversion of cytosine residues” refers to the conversion of cytosine residues to uracil residues as a result of bisulfite treatment.

“Exon” refers to a coding region of a genome.

“Hybridization conditions” refers to conditions that allow hybridization of complementary nucleic acid strands.

  “Nucleic acid sequencing” refers to determining the identity of at least one nucleotide of a target nucleic acid molecule, and in some embodiments, determining the identity of multiple nucleotides of a target nucleic acid molecule.

  “Immobilized” and “immobilize” refer to attaching nucleic acids to an individual support, directly or indirectly, by methods other than complementary base pairing. A specific hybridization complex is considered to be immobilized on an individual support as described above if at least one of the nucleic acid duplexes of a specific hybridization complex is immobilized on the individual support. It is.

  “Label” refers to any moiety that can be detected directly or indirectly.

  “Nucleic acid” refers to a polymer of nucleotides of any length.

  “Nucleotide” refers to nucleotides and analogs thereof that can be incorporated into a growing nucleic acid strand by a polymerase. Nucleotides include, but are not limited to, four types of nucleotides generally incorporated into DNA (adenine, guanine, cytosine and thymine), four types of nucleotides generally incorporated into RNA (adenine, guanine, cytosine and uracil), inosinic acid, etc. Includes nucleotides with modified bases and labeled or otherwise modified nucleotides.

  "Polymerase" refers to a natural or non-natural enzyme or enzymatically active fragment thereof that can incorporate nucleotides into a nucleic acid strand that extends under polymerization conditions, including but not limited to DNA polymerase, RNA polymerase, and reverse transcription. Contains enzymes.

  “Polymerization conditions” refers to conditions that allow a polymerase to incorporate nucleotides into a nucleic acid strand.

  “Primer” refers to a nucleic acid to which nucleotides can be added by a polymerase. “Additional” refers to the addition of nucleotides directly to a primer by a polymerase, and then the addition of nucleotides to the extending nucleic acid strand from the primer.

  “Priming-competent structure” refers to the primer having a reactive group that the polymerase can utilize for nucleotide addition.

  “Individual support” refers to any individual support.

  A “specific hybridization complex” refers to a specific hybridization complex that can be formed or substantially maintained under stringent hybridization conditions and / or stringent wash conditions.

  “Target nucleic acid molecule” refers to any target nucleic acid molecule of interest.

  “Template” refers to a denatured region of a single-stranded or double-stranded nucleic acid that can be used by a polymerase to synthesize a complementary nucleic acid strand.

(II. Capture and sequencing of target nucleic acid molecule)
In one embodiment, the invention relates to a method of capturing a target nucleic acid molecule using a complementary nucleic acid, such as a primer, immobilized on an individual support. In certain embodiments, the nucleic acid sequence of the target nucleic acid molecule is then determined by detecting nucleic acid polymerization from a primer by a polymerase using the target nucleic acid molecule as a template. Detection occurs at the single molecule level and is in real time or near real time.

(Target nucleic acid molecule)
In various embodiments, the target nucleic acid molecule is DNA. In one embodiment, the target nucleic acid molecule may correspond to any region of the genome (“target region”), such as the human genome or the genome of any other organism. A target region is known to contain one or more multi-megabase contiguous blocks, multiple smaller contiguous or non-contiguous regions, such as full exons of one or more chromosomes, or SNPs And so on. The genome containing the target region may be partial or complete. The genome can be any patient sample or pooled patient sample, cell line or cell culture medium, biopsy material, normal tissue sample or tumor or other diseased tissue and other biological sources recognized by those skilled in the art. From any biological source. In one embodiment, the genomic DNA containing the target nucleic acid is sheared, eg, by sonication or hydrodynamic forces, into generally about 200-600 base pair fragments, and the target nucleic acid molecule is a fragment or fraction thereof. Captured from. In another embodiment, the target nucleic acid molecule can be a coding sequence or a non-coding sequence. In one such embodiment, the target nucleic acid molecule is an exon or a part thereof.

  In various embodiments, the target nucleic acid molecule is RNA. In one embodiment, the target nucleic acid molecule is an mRNA transcript or a portion thereof. In one such embodiment, the target nucleic acid molecule is an mRNA transcript or a portion having a poly-A tail thereof. The presence of the poly-A tail generally allows hybridization to probes or primers that contain a sufficiently long poly-T sequence at the 3 'end of the primer. In another embodiment, the target nucleic acid molecule is a cDNA generated from RNA, such as by reverse transcriptase.

(capture)
In various embodiments, the target nucleic acid molecule is captured from a mixture of nucleic acids such as RNA, DNA (eg, genomic DNA) or cDNA molecules. In one embodiment, the nucleic acids in the mixture are amplified prior to capture of the target nucleic acid molecule. This can be obtained, for example, by ligating an adapter containing a universal priming site to the end of a nucleic acid molecule in the mixture, which can optionally be end-repaired prior to ligation. Universal primers can thus be used for nucleic acid amplification in a mixture.

  In various embodiments, the target nucleic acid molecule is captured using a complementary nucleic acid immobilized on an individual support. The complementary nucleic acid need not be completely complementary to the target nucleic acid molecule and may contain mismatches as long as the target nucleic acid molecule and the complementary nucleic acid molecule can form a specific hybridization complex. In one embodiment, the complementary nucleic acid is a primer. In one such embodiment, the primer is immobilized on the solid support in a priming capable structure. For example, a primer with 3'-OH is immobilized on a solid support and 3'-OH can be used by a polymerase to add nucleotides to the 3 'end of the primer. This can be obtained, for example, by immobilizing the primer to the solid support at its 5 'end, or at an internal region of the primer, so long as the primer is capable of priming. The primer can be of any length as long as it can form a specific hybridization complex with the target nucleic acid molecule, and in certain embodiments the primer length is at least 10, 15, 20, 25, 50, 75, 100, 200. 300, 400 or 500 base pairs.

  The art leads to methods for immobilizing nucleic acids to an individual support. For example, nucleic acids such as the complementary nucleic acids provided above can be immobilized to an individual support, such as covalently or non-covalently. Suitable chemical linkers or other attachment methods are known to those skilled in the art. For example, in one embodiment, the nucleic acid to be immobilized is biotinylated (eg, comprises one or more biotinylated nucleotides), the solid support has streptavidin on its surface, and the biotin portion of the nucleic acid is streptoid. It binds to avidin and immobilizes nucleic acid. In another embodiment, the immobilization process provided in the above embodiments is obtained by synthesizing nucleic acid on an individual support. For example, a primer can be synthesized on an individual support by polymerizing nucleotides in the 5 'to 3' direction, leaving 3'-OH available at the primer end distal to the individual support. Chemical methods for synthesizing oligonucleotides in the 5 'to 3' direction on a solid support, such as high density microarrays, are known in the art and can be used for the purposes described herein. See Albert et al. (2003) “Light directed 5 ′ → 3 ′ synthesis of complex oligonucleotide microarrays” Nucleic Acids Res. 31 (7): e35, which is incorporated herein by reference in its entirety.

  In various embodiments, the solid support can be any substrate on which the nucleic acid can be immobilized. Such substrates include but are not limited to glass (eg, microscope glass slides), metals, ceramics, polymeric beads and other substrates. In certain embodiments, the individual support may be in the form of an array, such as a microarray. In certain embodiments, the nucleic acid may be immobilized on an individual support, such as a bead, and then captured, or may be immobilized on another individual support, such as a glass slide or microarray.

  In certain embodiments, an individual support to which a complementary nucleic acid is immobilized is exposed to a mixture of nucleic acids comprising a target nucleic acid molecule under hybridizing conditions. The target nucleic acid molecule thus forms a specific hybridization complex with the complementary nucleic acid. In another embodiment, the individual support is washed to remove non-binding nucleic acids and non-specific binding nucleic acids, thereby allowing target nucleic acid molecules (contained in the specific hybridization complex) to be mixed with other nucleic acids in the mixture. Separate from nucleic acids. In certain embodiments, exposing the individual support to the nucleic acid mixture and / or washing the individual support is performed under stringent hybridization conditions and / or stringent wash conditions, respectively.

  As used herein, “hybridization” refers to pairing of complementary nucleic acid strands. Hybridization and its strength (ie, the strength of association between nucleic acid strands) are influenced by factors such as the degree of complementarity between nucleic acids, the stringency of conditions, the Tm of the hybridization complex, and the G: C ratio of the nucleic acid. Although the present invention is not limited to a specific set of hybridization conditions, stringent hybridization conditions may be used. Stringent hybridization conditions may be determined empirically by those skilled in the art using routine methods. Stringent hybridization conditions depend on the sequence as well as environmental factors such as salt concentration and the presence of organic solvents. Generally, stringent hybridization conditions are selected to be about 5 ° C. to 20 ° C. lower than the thermal melting point (Tm) of the specific nucleic acid sequence at a defined ionic strength and pH. In certain embodiments, stringent hybridization conditions are about 5 ° C. to 10 ° C. lower than the thermal melting point of a specific nucleic acid that binds to a complementary nucleic acid. Tm is the temperature at which 50% of a nucleic acid (eg, a target nucleic acid molecule) hybridizes (with a defined ionic strength and pH) to a perfectly compatible primer.

  Similarly, stringent wash conditions may be determined empirically by those skilled in the art using routine methods. For example, stringent washing conditions are conditions that can reliably separate non-specifically bound nucleic acids from a specific hybridization complex immobilized on an individual support such as an array. In one embodiment, the array is exposed to hybridization conditions (e.g., stringent hybridization conditions), followed by buffers and / or high concentrations of detergents and / or increasing temperatures that are successively reduced in concentration. The signal-to-noise ratio of specific hybridization to non-specific hybridization is high enough to easily detect specific hybridization, e.g. hybridization between nucleic acid strands sharing complete or substantially complete complementarity. It is washed until. In certain embodiments, stringent wash conditions comprise a temperature of about 30 ° C, 37 ° C, 42 ° C, 45 ° C, 50 ° C or 55 ° C. In certain embodiments, the salt concentration of stringent wash conditions comprises ≦ 1M, ≦ 500 mM, ≦ 250 mM, ≦ 100 mM, ≦ 50 mM or ≦ 25 mM, but not ≧ 10 mM. An example of stringent hybridization conditions is 50% formamide, 5 × SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5% X Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS and 10% dextran sulfate at 42 ° C. An example of stringent washing conditions is 55 ° C. in 0.1 × SSC (containing EDTA).

(Sequencing)
The target nucleic acid molecule (contained in the specific hybridization complex) can be sequenced after being separated from other nucleic acids in the mixture. In various embodiments, this step is commonly referred to as “resequencing” and the sequence of the target region from the reference genome is known. Current methods of resequencing require that the target nucleic acid molecule be amplified and then eluted from the specific hybridization complex, and the amplified target nucleic acid molecule is then subjected to “next generation” sequencing techniques (eg, in parallel). Sequencing is determined using sequencing platforms capable of high-throughput sequencing) or resequencing arrays (microarrays containing probes that specifically detect the presence or absence of mutations in individual segments of nucleic acids). The requirements of the elution and amplification steps are time and resource consuming and can lead to sample loss or bias indication of individual target nucleic acid molecules within the target nucleic acid molecule population.

  Thus, in various embodiments, the target nucleic acid molecule is resequenced without eluting the target nucleic acid molecule from the specific hybridization complex. This is achieved in one embodiment when the complementary nucleic acid contained in the specific hybridization complex is a primer immobilized on the solid support in a priming capable structure. For example, a primer can be hybridized to a specific region of a target nucleic acid molecule, with the remaining portion of the nucleic acid molecule being in single-stranded form. Thus, the polymerase can use the single stranded (non-hybridized) portion of the target nucleic acid molecule as a template to add nucleotides to the available 3'-OH of the primer. The nucleic acid strand synthesized by the polymerase is used to determine the sequence of the target nucleic acid molecule.

  Thus, the solid support to which the specific hybridization complex is immobilized through the primer can be exposed to the polymerization reaction mixture. In one embodiment, the polymerization reaction mixture comprises a polymerase and nucleotides. The polymerase may be a DNA polymerase, RNA polymerase or reverse transcriptase. If the target nucleic acid molecule is RNA, the polymerase can be reverse transcriptase. In another embodiment where the target nucleic acid molecule is DNA, the polymerase is a DNA polymerase. Some exemplary DNA polymerases include, but are not limited to, bacterial DNA polymerases (eg, E. coli DNA, pol I, II, III, IV, and V and the Klenow fragment of DNA pol I), viral DNA polymerases (eg, T4 and T7 DNA polymerase), archaeal DNA polymerase (eg, Thermus Aquatics (Taq) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, “Deep Vent” DNA polymerase (New England BioLabs), eukaryotic Biological DNA polymerases and their engineered or modified variants, etc. Some exemplary RNA polymerases include, but are not limited to, T7, T3 and SP6 RNA polymerases and their engineered or modified variants. Some exemplary reverse transcriptases include, but are not limited to, reverse transcriptases from HIV, MMLV and AMV and commercially available reverse transcriptases such as Superscript (Invitrogen, Carlsbad, Calif.).

  In certain embodiments, the nucleotides and / or polymerases in the polymerization reaction mixture are labeled. In one embodiment, 1, 2, 3 or 4 nucleotides are differentially labeled. In one such embodiment, the four nucleotides are labeled with four different labels. For example, in the case of dNTPs, adenine (or its functionally equivalent analog), guanine (or its functionally equivalent analog), cytosine (or its functionally equivalent analog) and thymine (or its functionally equivalent) Each) is labeled with a separate label, such as a different fluorophore. Similarly, in the case of rNTPs, adenine (or a functionally equivalent analog thereof), guanine (or a functionally equivalent analog thereof), cytosine (or a functionally equivalent analog thereof) and uracil (or a functionally equivalent thereof) Each equivalent analog) is labeled with a separate label, such as a different fluorophore. Suitable labels include, but are not limited to, luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent and / or phosphorescent labels. Fluorescent labels include, but are not limited to, xanthine dyes, fluorescein, cyanine, rhodamine, coumarin, acridine, Texas red dye, BODIPY, ALEXA, GFP and modifications thereof. The label may be attached directly to the nucleotide or via an appropriate linker. The label may be attached at any position that does not significantly interfere with the ability of the polymerase to incorporate nucleotides into the extending nucleic acid strand. In one embodiment, the label is attached to the phosphate of the nucleotide, eg, the phosphate at the end of the nucleotide, so that the phosphate chain of the nucleotide and the label are cleaved upon incorporation of the nucleotide into the extending nucleic acid chain. The labeled nucleotide and / or polymerase may allow detection of the nucleic acid strand synthesized by the polymerase.

  In one embodiment, the sequence of the target nucleic acid molecule is determined by identifying the nucleotides that are incorporated into the nucleic acid strand that is extended by the polymerase in the order in which they are incorporated. In one embodiment, the nucleic acid extending by detecting the label of the nucleotide incorporated into the extending nucleic acid strand, directly or indirectly, in the order in which the nucleotide is incorporated, and correlating the detected label with the nucleotide identity. Including confirming the sequence of the strands. Thus, the sequence of the target nucleic acid molecule (or its complementary strand depends on whether the primer hybridizes to the sense strand or the antisense strand of the target nucleic acid molecule). In such embodiments, the detection of the label and essentially the sequencing of the target nucleic acid molecule is performed at a real time and “single molecule” level. As described above and below, since the label is removed at the same time as the nucleotide is incorporated into the extending nucleic acid strand, the resulting nucleic acid strand is not labeled.

  Several methods for detecting labeled nucleotides incorporated into elongating nucleic acid strands are described in WO 2010/002939. Such a method relies on Forster resonance energy transfer between a “donor” molecule (FRET (Forster Resonance Energy Transfer) donor) and an “acceptor” molecule (FRET acceptor) when the molecules are at a distance close enough to the difference. . In a particular embodiment, the polymerase is labeled with a FRET donor fluorophore and the nucleotide is labeled with a FRET acceptor fluorophore. When the polymerase incorporates the nucleotide into the elongating nucleic acid strand, the FRET donor fluorophore and the FRET acceptor fluorophore are brought close together, allowing energy transfer from the FRET donor fluorophore to the FRET acceptor fluorophore. Due to the energy transfer, the emission intensity from the FRET donor fluorophore decreases and the emission intensity of the FRET acceptor fluorophore increases. Detection of the emission spectrum of the FRET acceptor indicates the identity of the incorporated nucleotide. In one embodiment, the FRET donor attached to the polymerase is a fluorescent nanoparticle, such as a nanocrystal, more specifically a quantum dot, as described in WO 2010/002939. The FRET donor may be irradiated with an excitation source such as a laser that generates donor emission. In another embodiment, different FRET acceptors are attached to each of one or more types of nucleotides, more specifically to each of three or four types of nucleotides. The FRET acceptor may be any of the fluorescent labels described above.

  The label can be detected using any suitable method or device such as, but not limited to, a charge coupled device and a total reflection microscope.

  In embodiments where the target nucleic acid molecule is poly-RNA, the target nucleic acid molecule is captured using a primer comprising a poly-T sequence. The cDNA is synthesized from the primer using reverse transcriptase (but not sequenced). The target nucleic acid molecule is then denatured from the specific hybridization complex of the individual support leaving a newly synthesized cDNA strand and immobilized to the individual support via a primer. The solid support is then exposed to a primer containing poly-A that hybridizes to the newly synthesized cDNA. The newly synthesized cDNA is sequenced by exposing the solid support to the polymerization reaction mixture as described above, and the DNA polymerase synthesizes the nucleic acid strand from the poly A primer.

  Using the method described above, a plurality of target nucleic acid molecules can be selected by selecting primers specific to each target nucleic acid molecule of interest and immobilizing the primers in individual areas of an individual support, such as a microarray, Can be separated and sequenced. In this way, target nucleic acid molecules of interest can be separated and sequenced with high throughput.

(III Determination of methylation status)
In another aspect, the present invention determines the methylation status of CpG dinucleotides in a genomic DNA fragment by immobilizing the genomic DNA fragment to an individual support, and the nucleic acid sequence of the immobilized genomic DNA fragment is determined as a genomic DNA fragment. Is determined by detection of nucleotide polymerization with a polymerase using as a template, the immobilized genomic DNA fragment is denatured from the newly synthesized nucleic acid strand, the individual support is exposed to bisulfite, and the nucleic acid sequence of the genomic DNA fragment Is determined by detection of nucleotide polymerization by a polymerase using a target nucleic acid molecule as a template.

(Genomic DNA fragment)
Genomic DNA fragments can be obtained from any genome, such as a human genome or a genome from any other organism. The genome may be partial or complete. The genome can be any patient sample or pooled patient sample, cell line or cell culture medium, biopsy material, normal tissue sample or tumor or other diseased tissue and other biological sources recognized by those skilled in the art. From any biological source. In one embodiment, genomic DNA is sheared, for example, by sonication or hydraulic forces, and is generally made into fragments of about 200-600 base pairs. In another embodiment, the genomic DNA is fragmented by enzymatic digestion.

(Fixation of genomic DNA fragment)
The genomic DNA fragment can be fixed to the individual support in any of a variety of ways. The genomic DNA fragment can be directly or indirectly fixed to the individual support, such as covalently or non-covalently. Suitable chemical linkers or other attachment methods are known to those skilled in the art. In certain embodiments, the genomic DNA fragment is denatured and fixed in a single-stranded form on an individual support. A genomic DNA fragment, for example, a single-stranded genomic DNA fragment, is fixed to an individual support via a binding nucleic acid or an adapter. For example, the adapter can be ligated to one or both ends of the genomic DNA fragment while being fixed to the solid support. The adapter can be single stranded, eg, an oligonucleotide. In some embodiments, the adapter is first ligated to the genomic DNA fragment and then fixed to the individual support, or the adapter is first fixed to the individual support and then the genomic fragment is then attached to the adapter on the individual support. Ligated. In another embodiment, the adapter is biotinylated (eg, comprises one or more biotinylated nucleotides), the solid support has streptavidin on its surface, and the biotin moiety binds to streptavidin and the adapter To fix.

  The direction of the fixed genomic DNA fragment may be 5 ′ → 3 ′ from the individual support or 3 ′ → 5 ′ from the individual support. In one embodiment, the immobilized genomic DNA fragment is directed 3 '→ 5' from the individual support. In another embodiment, the fixed genomic DNA fragment is single stranded. In another embodiment, the single stranded genomic fragment is immobilized on an individual support via an adapter, such as an oligonucleotide. In certain embodiments, the genomic DNA fragment is single stranded and ligated to an adapter fixed to the solid support, with the adapter and genomic DNA fragment being directed 3 '→ 5' from the solid support.

(Sequencing)
The sequence of the fixed genomic DNA fragment is determined on an individual support. In one embodiment, the genomic DNA fragment is fixed to the solid support in a single-stranded form, or fixed to the solid support in a double-stranded form, all or part of which is fixed to the solid support. It can be converted to a single strand as it is.

  In certain embodiments, an individual support is exposed to a primer under hybridization conditions, and the primer and genomic DNA fragment form a specific hybridization complex. In certain other embodiments, the individual support is exposed to the primer under hybridization conditions, and the primer and adapter form a specific hybridization complex. In one such embodiment, the adapter is an oligonucleotide to which a genomic DNA fragment is ligated and the adapter is immobilized on an individual support. In the foregoing embodiment, cytosine in the nucleic acid sequence to which the primer in the genomic DNA fragment or adapter binds is protected from deamination resulting from bisulfite treatment, for example by having a protecting group. The protecting group can be, for example, a methyl group, and the cytosine to be protected can be 5-methylcytosine.

  In another embodiment, the solid support is exposed to the polymerization reaction mixture. In one embodiment, the polymerization reaction mixture comprises DNA polymerase and nucleotides, which synthesize nucleic acid strands from the primers using genomic DNA fragments as templates. In one such embodiment, the DNA polymerase synthesizes a nucleic acid strand from primers that form a specific hybridization complex with the adapter, and the adapter (optional) and the genomic DNA fragment are used as a template. For example, if the adapter and genomic DNA fragment are oriented 3 ′ → 5 ′ from the individual support and the adapter binds the genomic DNA fragment to the individual support, the primer is 5 ′ → 3 ′ from the individual support. Because it can hybridize to the adapter in the direction, the primer primes the synthesis in the 5 ′ → 3 ′ direction using the adapter by the polymerase and (optionally) the genomic DNA fragment as a template. In another such embodiment, the DNA polymerase synthesizes a nucleic acid strand from a primer that forms a specific hybridization complex with the genomic DNA fragment, and the genomic DNA fragment is used as a template.

  Suitable DNA polymerases include, but are not limited to, bacterial DNA polymerases (eg, E. coli DNA, pol I, II, III, IV, and V and Kpolnow fragments of DNA pol I), viral DNA polymerases (eg, T4 and T7). DNA polymerase), archaeal DNA polymerase (eg, Thermus Aquatics (Taq) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, “Deep Vent” DNA polymerase (New England BioLabs), eukaryotic organism DNA polymerases and their engineered or modified mutants are included.

  In certain embodiments, the nucleotides and / or polymerases in the polymerization reaction mixture are labeled. In one embodiment, 1, 2, 3 or 4 nucleotides are differentially labeled. In one such embodiment, the four nucleotides are labeled with four different labels. For example, in the case of dNTPs, adenine (or its functionally equivalent analog), guanine (or its functionally equivalent analog), cytosine (or its functionally equivalent analog) and thymine (or its functionally equivalent) Each) is labeled with a separate label, such as a different fluorophore. Suitable labels include, but are not limited to, luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent and / or phosphorescent labels. Fluorescent labels include, but are not limited to, xanthine dye, fluorescein, cyanine, rhodamine, coumarin, acridine, Texas red dye, BODIPY, ALEXA, GFP and modifications thereof. The label may be attached directly to the nucleotide or via an appropriate linker. The label may be attached at any position that does not significantly interfere with the ability of the polymerase to incorporate nucleotides into the extending nucleic acid strand. In one embodiment, the label is attached to the phosphate of the nucleotide, eg, the phosphate at the end of the nucleotide, so that the phosphate chain of the nucleotide and the label are cleaved upon incorporation of the nucleotide into the extending nucleic acid chain. The labeled nucleotide and / or polymerase may allow detection of the nucleic acid strand synthesized by the polymerase.

  In one embodiment, the sequence of the genomic DNA fragment is determined by identifying the nucleotides that are incorporated into the nucleic acid strand that is extended by the polymerase in the order of incorporation. In one embodiment, the nucleic acid extending by detecting the label of the nucleotide incorporated into the extending nucleic acid strand, directly or indirectly, in the order in which the nucleotide is incorporated, and correlating the detected label with the nucleotide identity. Including confirming the sequence of the strands. Thus, the sequence of the genomic DNA fragment (or the complementary strand depending on whether the sense strand or the antisense strand of the genomic DNA fragment is fixed) is determined. In such an embodiment, the detection of the label and essentially the sequencing of the genomic DNA fragment is performed at a real time and “single molecule” level. As described above and below, since the label is removed at the same time as the nucleotide is incorporated into the extending nucleic acid chain, the newly obtained nucleic acid chain is not labeled.

  Several methods for detecting labeled nucleotides incorporated into elongating nucleic acid strands are described in WO 2010/002939. Such a method relies on Forster resonance energy transfer between a “donor” molecule (FRET (Forster Resonance Energy Transfer) donor) and an “acceptor” molecule (FRET acceptor) when the molecules are at a distance close enough to the difference. . In a particular embodiment, the polymerase is labeled with a FRET donor fluorophore and the nucleotide is labeled with a FRET acceptor fluorophore. When the polymerase incorporates the nucleotide into the elongating nucleic acid strand, the FRET donor fluorophore and the FRET acceptor fluorophore are brought close together, allowing energy transfer from the FRET donor fluorophore to the FRET acceptor fluorophore. Due to the energy transfer, the emission intensity from the FRET donor fluorophore decreases and the emission intensity of the FRET acceptor fluorophore increases. Detection of the emission spectrum of the FRET acceptor indicates the identity of the incorporated nucleotide. In one embodiment, the FRET donor attached to the polymerase is a fluorescent nanoparticle, such as a nanocrystal, more specifically a quantum dot, as described in WO 2010/002939. The FRET donor may be irradiated with an excitation source such as a laser that generates donor emission. In another embodiment, different FRET acceptors are attached to each of one or more types of nucleotides, more specifically to each of three or four types of nucleotides. The FRET acceptor may be any of the fluorescent labels described above.

  The label may be detected using any suitable method or device such as, but not limited to, a charge coupled device and a total reflection microscope.

  Using the methods described above, a plurality of genomic DNA fragments can be isolated and sequenced by immobilizing the genomic DNA fragments in individual sections of an individual support, such as a microarray. In this way, the genomic DNA fragment of interest can be fixed and sequenced with high throughput.

(Bisulfite treatment and sequencing after treatment)
Following sequencing of the genomic DNA fragment, the specific hybridization complex consisting of the newly synthesized DNA strand and the genomic DNA fragment is denatured (eg, by heat or base denaturation) and the newly synthesized DNA strand is Separated from DNA fragments. The genomic DNA fragment on the solid support is subjected to bisulfite treatment. Methods for performing bisulfite treatment are known in the art and are described, for example, in Herman et al. (1996) Proc. Natl. Acad. Sci. USA 93: 9821-9826. The unprotected (unmethylated) cytosine of the genomic DNA fragment is thus converted to uracil. The genomic DNA fragment is then subjected to a sequencing protocol as described above. The resulting sequence is compared with the sequence obtained prior to bisulfite treatment, as shown by the presence of thymine instead of guanine in the newly synthesized chain generated by the sequencing protocol, for example: A cytosine residue converted to a uracil residue in the genomic DNA fragment is identified. Residues that have been converted show an unmethylated state in the genomic DNA fragment, whereas residues that have not been converted show a protected, ie methylated state, in the genomic DNA fragment. In this way, the methylation state of the genomic DNA fragment is determined.

[a] Direct capture and real-time sequencing of DNA molecules DNA containing the target nucleic acid molecule of interest (eg, genomic DNA with the protein coding region of interest) is suitable as shown in FIGS. 1B and 2B Sheared to size. A substrate, such as an array (shown in FIG. 1A) or a bead (shown in FIG. 2A), containing a complementary oligonucleotide of the region of interest is used. The region of interest can be, for example, an exon. Nucleic acid containing the region of interest from the sheared DNA is captured on the substrate through hybridization to the oligonucleotide. In FIG. 1, nucleic acids containing the relevant regions from sheared DNA are captured on the array by hybridization to oligonucleotides. In FIG. 2, the nucleic acid containing the corresponding region from the sheared DNA is captured in the solution on the beads. Non-binding DNA and non-specific binding DNA are washed away. In FIG. 2C, the beads are placed on another substrate, such as a regular or irregular array. The captured DNA is subjected to direct sequencing as shown in FIGS. 1C and 2C. This is obtained using the free 3 ′ end of the oligonucleotide (acting as a primer), labeled polymerase and labeled dNTP that allow direct monitoring of the added base and polymerase during real-time DNA synthesis.

[b] Direct capture and real-time sequencing of RNA molecules RNA containing poly-A tails can be used, as shown in FIGS. 3B and 4B. Substrates such as, for example, arrays (shown in FIG. 3A) or beads (shown in FIG. 4A) comprising oligonucleotides comprising poly-dT are also used. RNA is captured on the substrate through hybridization of a poly-A tail to an oligonucleotide containing poly-dT, as shown in FIGS. 3C and 4C. Unbound RNA and non-specifically bound RNA are washed away and the captured RNA is converted to cDNA using the free 3 ′ end of poly-dT and reverse transcriptase. After conversion from RNA to cDNA, the cDNA sequence is determined. In one embodiment of cDNA sequencing, an oligonucleotide adapter is ligated to the free 3 ′ end of the newly synthesized cDNA, and then a primer is annealed to the adapter. The cDNA is sequenced using the primer, labeled polymerase and labeled dNTP, which allows direct monitoring of added base and polymerase at the single molecule level during real-time DNA synthesis. Alternatively, the captured RNA can be sequenced directly using labeled reverse transcriptase and labeled dNTPs that allow direct monitoring of added bases and polymerases at the single molecule level during real-time cDNA synthesis. Can be done. (See Figures 3C and 4C.)

[c] Methylation status determination by recursive sequencing of the same template To determine the methylation status of DNA (eg genomic DNA), the DNA is first sheared to the appropriate size, preferably as shown in FIGS. 5B and 6B. Converted to single strand as shown. Substrates such as arrays (shown in FIG. 5A) or beads (shown in FIG. 6A) are used, including methylated oligonucleotides (ie, oligonucleotides containing methylcytosine). Sheared DNA is ligated to a methylated oligonucleotide as shown in FIGS. 5C and 6C. The ligated DNA then has complementary primers for the methylated oligonucleotides on the array or bead, labeled polymerase, and direct monitoring of the added base and polymerase at the unimolecular level during real-time DNA synthesis. Sequencing is performed using labeled dNTPs. After sequencing the ligated DNA, the newly synthesized strand is removed and the original DNA is bisulphite so that uracil conversion of methylated cytosine is possible, as shown in FIGS. 5D and 6D. It is processed. The treated DNA is sequenced using primers, labeled polymerase and labeled dNTP that allow direct monitoring of added bases and polymerase at the unimolecular level during real-time DNA synthesis. By comparing sequences before and after bisulfite treatment obtained from the same molecule, the methylation state of DNA can be determined.

  All patents and publications cited within this specification are incorporated by reference.

Claims (16)

A method for capturing and sequencing a target nucleic acid molecule comprising:
(A) exposing an individual support to a mixture of nucleic acids comprising a target nucleic acid molecule under hybridizing conditions, wherein the target nucleic acid molecule is specific to a primer immobilized on the individual support in a priming capable structure Forming a hybridization complex,
(B) separating non-binding nucleic acid and non-specific binding nucleic acid from the solid support,
(C) exposing the solid support to a polymerase and nucleotides under polymerization conditions;
(D) determining the nucleic acid sequence of the target nucleic acid molecule by detecting nucleic acid polymerization by a polymerase using the target nucleic acid molecule as a template from immobilized primers.   The method of claim 1, wherein the target nucleic acid molecule is derived from a genomic DNA region.   The method of claim 2, wherein the target nucleic acid molecule comprises all or part of an exon.   The method of claim 1, wherein the target nucleic acid molecule is RNA, the polymerase is reverse transcriptase, and the primer comprises a 3 'poly-T sequence.   The method according to claim 1, wherein the target nucleic acid molecule is DNA and the polymerase is a DNA polymerase.   The method of claim 1, wherein the nucleotide is labeled to a terminal phosphate.   The method of claim 6, wherein the polymerase is labeled with a FRET donor and the nucleotide is labeled with a FRET acceptor.   8. The method of claim 7, wherein the FRET donor is a fluorescent nanoparticle. A method for determining the methylation status of a genomic DNA fragment comprising:
(A) fixing a genomic DNA fragment to an individual support;
(B) determining the nucleic acid sequence of a genomic DNA fragment immobilized on an individual support by detecting nucleic acid polymerization by a polymerase using the immobilized genomic DNA fragment as a template;
(C) subjecting the fixed genomic DNA fragment to bisulfite treatment;
(D) determining the nucleic acid sequence of the immobilized bisulfite-treated genomic DNA fragment by detecting nucleic acid polymerization by a polymerase using the immobilized bisulfite-treated genomic DNA fragment as a template;
(E) comparing the nucleic acid sequence determined in (b) with the sequence determined in (d), where the conversion of cytosine residues in the genomic DNA fragment is methylated prior to bisulfite treatment A method wherein the cytosine residue in the genomic DNA fragment is not converted, indicating that the residue was methylated prior to bisulfite treatment.   The method according to claim 9, wherein the genomic DNA fragment is fixed to the individual support by an adapter.   11. The method of claim 10, wherein the adapter comprises a primer binding site and cytosine in the primer binding site is protected.   12. The method of claim 11, wherein the polymerase of (b) and / or (d) polymerizes the nucleic acid strand from a primer annealed to the primer binding site.   10. The method of claim 9, wherein the nucleic acid polymerization of (b) and / or (d) is detected by detecting the incorporation of labeled nucleotides.   14. The method of claim 13, wherein the labeled nucleotide is labeled with a terminal phosphate.   15. The method of claim 14, wherein the polymerase of (b) and / or (d) is labeled with a FRET donor and the nucleotide is labeled with a FRET acceptor.   The method of claim 15, wherein the FRET donor is a fluorescent nanoparticle.

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